This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.

두 A. 1. 유생 양육 albopictus 그룹은 성인기 <ol><li> 두 최적의 휴면 식 (16)에 대해 21 ° C에서 프로그램 조명 광주 캐비닛과 약 80 %의 상대 습도를 설정합니다. <ol><li> 16L를위한 프로그램 하나 캐비닛 : 8D 등 : 어두운주기 (비 휴면는 LD의 광주를 유도). 8L의 두 번째 캐비닛을 설정합니다 16D 빛 : 어두운주기 (유도 휴면) 13. </li><li> 모두 캐비닛에 동시에 프로그램 &#39;에 조명&#39;photoperiods 사이의 일주기 시간을 동기화합니다. </li></ol></li><li> 실험을 수행하는 데 필요한 계란의 금액을 계산한다. 케이지 당 300 ~ 500 달걀을 목표로하고 있습니다. 세 적어도 휴면 케이지를 복제하고 세 비 휴면 케이지가 RNA 생성에 필요한 복제합니다. 이 실험 당 1,800-3,000 계란을시켜 대략 합계. <ol><li> 약에 탈 H 2 O의 500 ml에 계란 논문을 잠수하여 해치 계란 </li><li> 추가 &lt;EM&gt; 캘리포니아. 지상 개 음식과 소금물 새우로 구성된 1 ml의 음식 슬러리는 이전에 24 설명했다. 메쉬 컨테이너를 커버하고, 고무 밴드와 장소에 메쉬를 유지합니다. </li><li> 약 24 시간 동안 LD의 광주 캐비닛에 넣습니다. </li></ol></li><li> 전송 부화 유충에 10 × 10 × 2cm 페트리 약 90 ml로 채워진 요리 탈 H 2 O <ol><li> 접시 당 약 30 유충을 유지한다. 예를 들어, 매주 월요일, 수요일, 금요일 (MWF) (24) 요리마다 48 ~ 72 시간을 청소하는 유충을 전송합니다. </li><li> 이전으로 탈 이온수에 지상 개 음식과 소금물 새우의 모든 MWF를 구성 피드 약 1 ml의 음식 슬러리 (24)을 설명했다. </li></ol></li><li> 각 케이지는 생물 복제를 포함하는 각각의 광주 처리를 위해 3-4 성인 케이지를 설정합니다. <ol><li> 9.5 L 버킷의 반대편에서 한 10 별 14cm의 구멍을 잘라, 그리고 15cm 직경이 다른 구멍. 공동메쉬와 첫 VER. 정형 스타킹의 약 다리 길이를 절단하고, 다른 구멍의 주위에 내측 일단 접착제. </li><li> 스타킹 오프 매듭 차단의 발 끝을 잘라 - 케이지의 내부에 대한 액세스가 필요한 경우에만 열립니다. 케이지 덮개를 들어, 만 림을 떠나, 내부를 모두 잘라, 메쉬 (24) 내부 플라스틱을 대체합니다. </li><li> 케이지의 측면에 영구 마커 실험에 관련된 번호, 케이지 시작 날짜 및 기타 정보를 복제, 광주를합니다. </li></ol></li><li> 젖은 종이 필터와 성인 케이지의 하단 라인. 종이 필터 (24)에 산란을 자극 할 수있는, 새장 현지 습도를 증가하지만, 고인 물을 피하기 위해 충분히 탈 H 2 O와 여과지를 적 십니다. 필요한 경우, 건조 매일 다시 젖은 여과지를 확인합니다. </li><li> 적어도 100 명의 여성을 포함, RNA 라이브러리에 대한 충분한 계란을 생산 / 9.5 L의 캘리포니아GE, 케이지 당 500 개 이상의 모기와. </li><li> 더 이상 50 번데기 (25) 당 ML의 H 2 O의 밀도로 깨끗한 H 2 O의 작은 컵에 번데기의 MWF과 장소를 수집 성인 케이지에 번데기 컵을 전송합니다. 각각의 광주 캐비닛에 넣어 케이지 - A. albopictus의 번데기는 15 감광 있습니다. </li><li> 죽은 번데기의 축적 대량 사망을 일으킬 수 있기 때문에, 컵에서 H 2 O는 맑고 깨끗한 있는지 매일 확인하고 죽은 번데기를 제거합니다. 모든 번데기가 등장 O 컵 후 H 2를 제거합니다. </li><li> 등장 성인 설탕을 제공하는 케이지의 상단 메쉬 유기농 건포도를 놓습니다. 건포도를 모니터링하고, 금형 축적을 방지하기 위해 모든 3~5일을 변경합니다. </li></ol> 성인 2. 유지 보수 상대와 계란 생산을 허용하는 <ol><li> (섹션에 ì을 습윤 여과지로 케이지 바닥을 안감으로 높은 습도 (약. 80 %)에서 케이지를 유지하고, 전술 한 바와 같이, 비 곰팡이가 핀 건포도에 대한 액세스를 제공기능 1.5 및 1.9). </li></ol> 3. 혈액 공급 <ol><li> 산란은 거의 100 % 휴면 계란 14 시작하기 전에 여성은 적어도 8 모호 짧은 일에 노출되었는지 확인하기 위해 우화 후 2-6일 사이의 혈액 공급 여성을 준비합니다. </li><li> Hemotek 막 먹이 시스템을 준비합니다. 전원 공급 장치에 공급 장치를 연결합니다. 조정 나사를 사용하여 37 ° C에 각 부의 온도를 조절합니다. 교정시 급지 유닛의 온도를 측정하기 위해 전자 온도계 및 프로브를 사용한다. </li><li> 식사 저수지를 준비합니다. 식사 저수지의 구멍을 통해 콜라겐 공급 막의 광장 스트레칭과 O 링으로 고정합니다. 조심스럽게 주름을 제거하기 위해 모서리를 당겨; 가위로 초과 막 트림. 참고 : 콜라겐 멤브레인은 모든 모기 종에 대해 잘 작동하지 않을 수 있습니다, 그리고 작업하는 경우 최적의 멤브레인을 찾기 위해 여러 종류를 시도 할 필요가있다A. 종 이외 albopictus. 파라 필름은 쿨 렉스 pipiens 잘 작동합니다. <ol><li> 혈액을 사용하기 전에 최소한 1 시간 동안 실온에서 동결, 해동 저장되어있는 경우. </li><li> 멤브레인이 아래를 향하도록 용기를 잡고, 지원되지 않는 및 충전 포트는 최대 직면하고있다. 항응고제로서 시트르산 나트륨을 갖는 닭에서 전혈 약 3 ml로 저장조를 채우기 위해 전송 피펫 또는 주사기를 사용한다. 플라스틱 플러그와 충전 포트를 밀봉합니다. </li></ol></li><li> 공급 장치 하단의 열 전달 접시에 스터드에 나사를 조이기에 의해 공급에 준비 저수지를 연결합니다. 모기는 케이지의 메쉬를 통해 공급할 수 있도록 공급 전환과 케이지의 위, 아래로 막면에 놓습니다. 공급을 최대화하기 위해 약 45 분 동안 케이지의 공급을 유지합니다. </li></ol> 4. 산란을 자극 <ol><li> 4 ~ 5 일 컬러 어두운 각 케이지를 장착, 혈액 식사를 게시 50 ㎖의 C표백 종자 발아 용지 (계란 용지) 또는 질감 비 표백 된 종이 타월로 줄 지어 탈 이온수 9 반쯤 채우까지. 250 개 이상의 모기가 케이지에있는 경우, 두 컵을 사용합니다. 참고 : 작은 케이지 또는 단일 여성 병의 경우, 산란 컨테이너에서 &quot;건초 주입은&quot;인해 미생물 식물 (25)의 냄새에 산란을 증가시킬 수있다. </li></ol> 5. 수집 및 저장 계란 <ol><li> 계란 생산은 일반적으로 다음 주에 걸쳐 진정 후 오일 혈액 공급 후 약 봉우리, 그리고 있기 때문에 혈액 공급 4 ~ 5 일 이내에 달걀 수집을 개시합니다. </li><li> 실험의 필요성에 달걀 수집 주파수를 다양합니다. 일반 목적을 위해, MWF 일정에 계란 서류를 수집합니다. 각 케이지에서 계란 서류를 제거하고 새 용지로 교체합니다. 계란 저장의 혼란 효과를 방지하기 위해 SD의 광주 캐비닛의 배양 접시와 저장소에 최근에 제거 서류를 놓습니다. </li><li> 계란 논문을 다시 허용<html> 계란 건조에 저항 (26)을 증가 장막 표피 형성을 허용하는 후 산란 2 일간 젖은 주. </li><li> 약 48 시간 게시물 수집, 야외에서 건조 계란. 이 논문은 H 2 O에서 어두운 또는 계란의 부화 자극하는 너무 젖은 다리를 저는 터치에 물기가 약간 있지만되도록 용지를 건조. 참고 : 6.5 &quot;× 4&quot;종이 건조하는 데 약 3.5 시간이 걸릴 수 있습니다. 이 계란 건조로 (27)가 발생합니다으로, 끝나지 건조 계란 논문에주의해야합니다. </li><li> LD와 SD 두 photoperiods에서 예약 추가 계란 휴면 발생률을 평가하고 (휴면 측정 제 6 장 참조) photoperiodic 효과를 해석합니다. </li><li> 장기 저장을 위해 21 ° C에서 계란 논문을 유지하고 페트리 접시에 약 80 % 습도. 배아 발달는 21 ° C에서 4~5일 사용되기 때문에 지역의 습도를 유지하기 위해 물을 플라스크와 유리 섬유 복합 재질의 저장 용기에서 배양 접시를 유지합니다. </li></ol>ove_title &quot;&gt; 6. 휴면 부각 측정 <ol><li> 추가 예약 배아 휴면 응답을 정량화하기 7-20일 나이 (산란, 4 장을 자극 참조)를 사용합니다. </li><li> 각 계란 종이에있는 계란의 수를 기록한다. </li><li> 완전히 약 80 ml의 탈 H 2 O와 함께 90 ml의 배양 접시에서 개별 계란 논문을 잠수하여 부화 달걀을 자극 약 0.25 ml의 음식 슬러리를 추가합니다. </li><li> 24 시간 후에 부화 첫번째 령 유충의 수를 집계. 애벌레를 시각화하고 접시의 한면에 광원을 배치하는 검은 색 표면에 페트리 접시를 놓습니다. 유충은 각각의 애벌레의 명확한 집계를 허용, 광원으로부터 멀리 이동합니다. 개별 유충을 열거 방지하기 위해 계산하는 동안 피펫 개별 유충을 제거합니다. </li><li> 새로운 배양 접시 및 재 건조에 넣어 달걀 논문입니다. 다시 해치 계란은 다시 1 ~ 일주일 후 위의 방법을 사용하여 부화 달걀을 집계. </li><li> 나머지 U와 장소 달걀 논문<html> 약 80 ml의 표백 액 28 ml의 90 새로운 배양 접시에 달걀을 N-화. 계란 논문이 완전히 표백 용액에 침수되어 있는지 확인하고 표백제 냄새를 방지하기 위해 흄 후드하에 밤새 둡니다. 주 : 표백 용액을 39 ° C에서 1 주간 ~ 저장 될 수 있지만, 그렇지 않은 신선한 이루어져야한다. </li><li> 융모막을 취소하고 발육, 취소 부화 달걀의 시각화를 허용 할 표백 광학 현미경을 이용하여 계란을 검사합니다. 난자가 발육 경우, 계란 등의 측면에서 서로 반대 두 개의 작은 검은 점으로 나타나는 눈 오프 화이트 색상이있을 것이다. 해칭되지 않은, 유정란 13의 수를 집계. </li><li> 없음 = %의 휴면 다음 식에 휴면 발생률을 결정합니다. 인간 백신 않은 부화 달걀 / (NO. 부화 달걀 + NO. 인간 백신 않은 부화 계란) (100) (13) X. </li></ol> 계란 / pharate 애벌레 7. RNA 추출 &quot;ontent&gt; 참고 : 층류 후드에서 사용 트리 졸. <ol><li> 낙타 털 브러시를 사용하여 유리 분쇄기에 달걀 논문에서 별개의 개발 시점에서 개발 배아 또는 pharate 유충을 포함하는 브러시 모기 계란. 완전히 분쇄 될 때까지 트리 졸에서 계란 (조직의 50 ~ 100 mg의 당 1 ml)에 갈기. 충분한 RNA를 생성하는 라이브러리 당 적어도 400 알을 사용합니다. <ol><li> 또한, 액체 질소에 동결 계란을 스냅 트리 졸 분쇄하기 전에 한 달까지를위한 마이크로 원심 튜브에서 -80 ° C에 저장됩니다. </li></ol></li><li> 트리 졸에 RNA 추출을 수행은 제조업체의 지시에 따라 이소프로판올 침전 하였다. </li><li> RNA 저하를 방지하기 위해 잔류 클레아 제를 제거하는 RNase 오염 제거 용액 또는 다른 에이전트와 벤치를 처리합니다. <ol><li> DNase의와 추출 된 RNA를 취급합니다. 제조업체의 지시에 따라, 37 ° C에서 30 분 동안의 DNase와 RNA 샘플을 배양한다. 1 #을 사용하여181; 50 μL 반응에서 RNA의 최대 10 μg의에 대한 리터의 DNase. 하나의 반응으로 RNA의 10 μg의가있는 경우의 DNase의 양을 늘립니다. </li><li> 5 μL 현탁 DNase의 불활 화 시약을 첨가하여 DNase의 불활. 잠복기 (부드러운 와동) 중 세 번을 혼합, 실온에서 5 분 품어. </li><li> 1.5 분 동안 10,000 XG에 원심 분리기. 후속 단계에 대한 신선한 튜브로 처리 된 RNA 샘플을 포함하는 상층 액을 전송합니다. </li></ol></li><li> 형광 측정에 의해 총 RNA 샘플의 품질을 평가. 이 작업에 대한 적절한 장비와 전문 시설로 샘플을 보낼 수 있습니다. 시설은 칩에 주입 형광 색소로 가시화 샘플 RNA 종의 크기를 결정하기 위해 온 - 칩 전기 영동을 수행한다. 결과는 electropherogram로 반환됩니다. <ol><li> 프리젠 O를 보면 알 수있는 바와 같이, 존재 또는 분해 생성물의 유무에 의해 총 RNA 샘플들의 무결성을 결정결과 electropherogram (그림 1B)의 18S와 5S 리보솜 RNA의 피크 사이의 F 봉우리. </li></ol></li></ol> 8. RNA 시퀀싱 <ol><li> 표준 프로토콜 다음 농축 쌍 엔드 mRNA의 라이브러리와 mRNA의 염기 서열의 건설을위한 상업 시퀀싱 센터에 충분히 높은 품질 (그림 1A)과 양 (라이브러리 당 보통&gt; 3 μg의)와 함께 총 RNA 샘플을 보낼 수 있습니다. </li><li> 둘 이상의 레인 하나를 시퀀싱 실험에 사용되는 경우, 시퀀스 동안 레인 기술 중 변동을 고려하여 시퀀싱 개의 레인으로 개인 라이브러리를 분할. </li></ol> 9. lllumina 읽기 청소 참고 : 그림 2는이 프로토콜의 생물 정보학 부분을 요약 한 것입니다. 이 프로토콜의 생물 정보학 섹션에 사용 된 모든 프로그램과 자료의 전체 목록은 표 1을 참조하십시오.에또한, 보조 파일 (1)는 다음과 같은 생물 정보학 프로토콜의 각 단계의 명령 줄 예제가 들어 있습니다. <ol><li> NCBI UniVec 코어 데이터베이스에 95 % 이상의 신원을 식별하기 위해 일치 ssaha2 29 (표 1)을 사용하여 (표 1), A. albopictus rRNA 유전자 서열 (GenBank 등록 번호의 L22060.1), 시퀀싱 어댑터 (보충 파일 1에서 제공하는 상세한 명령 줄 예제). 제공되는 Perl 스크립트 (보충 파일 2)를 적용하여, 예를 들어 펄을 사용하여 일치 또는 유사한 스크립트 도구와 읽기 쌍을 제거합니다. </li><li> 나머지 청소 SolexaQA 패키지 (30)와 읽기 (표 1, 보충 파일 1) : DynamicTrim.pl의 기본 설정을 사용하여 20 미만의 phred 점수 동등한 영역을 트림. <ol><li> 제거는 앞으로 모두 LengthSort.pl와보다 짧은 25 BP를 읽고 역을 동시에 읽습니다. FastQC (표 1 fastq 세정 파일의 품질을 평가 </st룽&gt;) - 특히, 당 염기 서열의 품질과 당 서열 품질 평가 점수가 20 위에 있는지 확인합니다. </li></ol></li></ol> 10. 디지털 정규화 <ol><li> K-메르 크기 20, 20의 범위 차단하고, X를 사용 (특히 normalize-by-median.py, (보충 파일 1 표 1) 크메르 도구 (31)를 사용하여 읽어 청소에 디지털 정상화 한 라운드를 수행 = 1E10). </li><li> 높은 RAM을 가진 기계를 사용할 수 (GB를 수백) 인 경우 또는, 삼위 일체의 normalize_by_kmer_coverage.pl 스크립트 (표 1)를 사용합니다. </li></ol> 11. 드 노보 사체 조립 <ol><li> 어셈블리의 크기에 따라, RAM의 최대 256 기가 24 CPU가있는 컴퓨터 또는 컴퓨터 클러스터에 대한 액세스 권한을 획득 할 수 있습니다. </li><li> 콘티로 설정 디지털 표준화 된 읽기를 조립; 삼위 일체 (32) (보충 파일 1 표 1)를 사용합니다. 줄이기 위해메모리 사용, --min_kmer_cov 2를 사용합니다. </li></ol> 12. 조립 평가 <ol><li> 삼위 일체 인접 출력에 Assemblathon2 프로젝트 (33)로부터 assemblathon_stats.pl를 실행합니다. (; 기업 파일 1 표 1)이 스크립트는 비계의 개수, N50, 어셈블리 조성물 등과 같은 조립 품질 평가에 관련된 기본적인 계산을 수행한다. </li></ol> 조립 사체 13. 주석 <ol><li> 기준 집합에 대해 단백질의 조립체 BLASTX (표 1)을 수행; 모기, 초파리,과 아노 펠 레스 gambiae, (Culex pipiens), 큘 및 Aedes aegypti 적합한 참조이다. 특히, BLASTX (보충 파일 1) 다음에 폭발에 대한 기준 단백질 FASTA 파일을 포맷합니다. </li></ol> (14)지도 RSEM (34)를 사용하여 총회에 읽습니다 (표 1) <ol><li> &#39;성적 증명서 - 투 - 유전자지도&#39;파일을 생성하는에첫 번째 열은 참조 유전자 ID 및 두 번째 열에 인접 ID를 포함합니다. 스프레드 편집기에서 BLASTX 출력으로부터 제 1 및 제 2 열을 교환하고,이 .txt 파일에 이러한 열 물품. 유닉스 형식으로 결과 .txt 파일에 줄 바꿈을 변환 할 LINEBREAK 프로그램을 사용하십시오. </li><li> RSEM 패키지 (보조 파일 1)에서 제공하는 rsem-준비 참조 스크립트를 사용하여 사체의 FASTA 파일에서 참조 데이터 집합을 만듭니다. </li><li> RSEM 패키지 (보조 파일 1)에 구비 rsem 계산할 표현식 명령을 이용하여 각각의 라이브러리에 대해 개별적 식 값을 계산한다. 읽을 때, 읽기 세척 단계 (단계 9.2)으로 인한 fastq 쌍 파일을 사용합니다. <ol><li> 생체로부터 RNA 복제가 시퀀싱 개의 레인으로 분리 된 경우, 하나의 파일을 생성하는 연산 식 모두 fastq 파일을 포함한다. </li></ol></li><li> 쉽게 다른 페이지에서 처리 행렬에 각 도서관에서 발현 결과를 변환RSEM 패키지 (보조 파일 1)에 설치된 설치 스크립트 rsem - 생성 - 매트릭스 데이터를 사용 rograms. </li></ol> 15. 차등 발현 분석 <ol><li> R 및 에저 (표 1)를 설치합니다. </li><li> 단계 14.3 (보충 파일 1)에서 RSEM 결과를로드 할 수 read.delim를 사용합니다. 필요한 경우, 가장 가까운 정수로 반올림 카운트. 참고 : EDGER 가이드는 중요성을 감지 충분히 높은 발현과 유전자 데이터 집합을 제한하는 것이 좋습니다. </li><li> EDGER에 대한 데이터 서식을 지정하려면로드 된 데이터 파일 (보충 파일 1)에서 DGEList 객체를 생성합니다. 이어서, TMM 정규화 (보조 파일 1)를 사용하여 데이터를 정상화. 데이터 (보충 파일 1)의 공통 및 tagwise 분산을 추정합니다. </li><li> Benjamini - 호흐 베르크로 발현 된 유전자를 확인하고 p- 값 &lt;0.05 (보충 파일 1) 수정. 풍부한 대 로그 배 변화 (보충 파일 1)의 분포를 그린다. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+adaptive+evolution+of+photoperiodic+response+during+invasion+and+range+expansion+across+a+climatic+gradient.">Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient.</a> Am. Nat. 179, 490-500 (2012).</li><li>Lounibos, L. P., Escher, R. L., Lourenco-de-Oliveria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+evolution+of+photoperiodic+diapause+in+temperate+and+tropical+invasive+populations+of+Aedes+albopictus+(Diptera+Culicidae).">Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera Culicidae).</a> Ann. Entomol. Soc. Am. 96, 512-518 (2003).</li><li>Mori, A., Oda, T., Wada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studies on the egg diapause and overwintering of Aedes albopictus in Nagasaki. Trop. Med. 23, 79-90 (1981).</li><li>Pumpuni, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors influencing photoperiodic control of egg diapause in Aedes albopictus [dissertation]. , (1989).</li><li>Wang, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+influence+of+photoperiod+on+egg+diapause+in+Aedes+albopictus+Skuse.">Observations on the influence of photoperiod on egg diapause in Aedes albopictus Skuse.</a> Acta Entomol. Sinica. 15, 75-77 (1966).</li><li>Sota, T., Mogi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival-time+and+resistance+to+desiccation+of+diapause+and+non-diapause+eggs+of+temperate+Aedes+(Stegomyia)+mosquitoes.">Survival-time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes.</a> Entomol. Exp. Appl. 63, 155-161 (1992).</li><li>Reynolds, J. A., Poelchau, M. F., Rahman, Z., Armbruster, P. A., Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+profiling+reveals+mechanisms+for+lipid+conservation+during+diapause+in+the+mosquito,+Aedes+albopictus.">Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.</a> J. Insect Physiol. 58, 966-973 (2012).</li><li>Andrewartha, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+in+relation+to+the+ecology+of+insects.">Diapause in relation to the ecology of insects.</a> Biol. Rev. 27, 50-107 (1952).</li><li>Danks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Dormancy:+An+Ecological+Perspective.">Insect Dormancy: An Ecological Perspective.</a> Biological Survey of Canada (Terrestrial Arthropods). , (1987).</li><li>Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+diapause.">Regulation of diapause.</a> Ann. Rev. Entomol. 47, 93-122 (2002).</li><li>Rev Entomol, A. n. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 59, 93-122 (2014).</li><li>Armbruster, P. A., Conn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+of+larval+growth+in+North+American+Aedes+albopictus+(Diptera).">Geographic variation of larval growth in North American Aedes albopictus (Diptera).</a> Culicidae). Ann. Entomol. Soc. Am. 99, 1234-1243 (2006).</li><li>Reiter, P., Amador, M. A., Colon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+CDC+ovitrap+with+hay+infusions+for+daily+monitoring+of+Aedes+aegypti+populations.">Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.</a> J. Am. Mosquito Contr. Association. 7 (1), 52 (1991).</li><li>Rezende, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+desiccation+resistance+in+Aedes+aegypti:+presumptive+role+of+the+chitinized+serosal+cuticle.">Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.</a> BMC Dev. Biol. 8, 182 (2008).</li><li>Munstermann, L., Crampton, J., Beard, C., C, L. o. u. i. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+and+maintenance+of+Aedes+mosquito+colonies.">Care and maintenance of Aedes mosquito colonies.</a> The Molecular Biology of Insect Disease Vectors. , 13-20 (1997).</li><li>Trpis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bleaching+and+decalcifying+method+for+general+use+in+zoology.">A new bleaching and decalcifying method for general use in zoology.</a> Can. J. Zoolog. 48, 892-893 (1970).</li><li>Ning, Z. M., Cox, A. J., Mullikin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSAHA:+A+fast+search+method+for+large+DNA+databases.">SSAHA: A fast search method for large DNA databases.</a> Genome Res. 11 (10), 1725-1729 (2001).</li><li>Cox, M. P., Peterson, D. A., Biggs, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SolexaQA:+At-a-glance+quality+assessment+of+Illumina+second-generation+sequencing+data.">SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data.</a> BMC Bioinformatics. 11, 485 (2010).</li><li>Grabherr, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nat. Biotechnol. 29 (7), 644-652 (2011).</li><li>Bradnam, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblathon+2:+evaluating+de+novo+methods+of+genome+assembly+in+three+vertebrate+species.">Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.</a> GigaScience. 2, 10 (2013).</li><li>Li, B., Dewey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>White, B. N., De Lucca, F. L., Turner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+RNA.">Preparation and analysis of RNA.</a> Analytical Biochemistry of Insects. , (1977).</li><li>Hawley, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Aedes+albopictus.">The biology of Aedes albopictus.</a> J. Am. Mosq. Contr. Assoc. 4, 1-39 (1988).</li><li>Dowling, Z., Ladeau, S. L., Armbruster, P., Biehler, D., Leisnham, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socioeconomic+status+affect+mosquito+(Diptera:Culicidae)+larval+habitat+type+availability+and+infestation+level.">Socioeconomic status affect mosquito (Diptera:Culicidae) larval habitat type availability and infestation level.</a> J. Med Entomol. 50, 764-772 (2013).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2,4,10.+Bloodfeeding:+Membrane+apparatuses+and+animals.">Chapter 2,4,10. Bloodfeeding: Membrane apparatuses and animals.</a> Methods in Anopheles Research. , (2010).</li><li>Das, S., Garver, S., Ramirez, J. R., Xi, Z., Dimopolous, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dengue+infections+in+mosquitoes+(A.+aegypti)+and+infection+phenotype+determination.">Protocol for dengue infections in mosquitoes (A. aegypti) and infection phenotype determination.</a> J. Vis. Exp. 5 (220), (2007).</li><li>Goff, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iPlant+collaborative:+cyberinfrastructure+for+plant+biology.">The iPlant collaborative: cyberinfrastructure for plant biology.</a> Plant Sci. 2 (34), (2011).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26, 139-140 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. 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The authors have nothing to disclose.

이 프로토콜은 A의 유도 휴면를 photoperiodically로 인해 발현 된 유전자를 발견하는 방법을 제시한다 albopictus. photoperiodic 휴면 응답에 초점을 사람들을 위해 특히 - 프로토콜은 고유 초보 사용자가 액세스 할 수있는 분자 생리학 프로그램의 모든 실험적인 측면을 만들기 위해 모기 양육과 생물 정보학 기술을 결합한다는 점에서 중요하다. 양육 실수를 파악하는 것이 필요 - - 기존의 방법...

Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1&#8211;3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8&#8211;10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15&#8211;17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Incubator - Model 818</td>
			<td style="width:116px;">Thermo-Scientific</td>
			<td align="right" style="width:109px;">3751</td>
			<td style="width:351px;">120V</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Controlled environment room</td>
			<td>Thermax Scientific</td>
			<td style="width:109px;">N/A</td>
			<td style="width:351px;">Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Cool Fluorescent bulb</td>
			<td style="width:116px;">Philips</td>
			<td align="right">392183</td>
			<td style="width:351px;">4 Watt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Petri Dish 100mm x 20mm</td>
			<td style="width:116px;">Fisher</td>
			<td>08-772-E</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Filter Paper 20.5cm</td>
			<td style="width:116px;">Fisher</td>
			<td>09-803-6J</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">9.5L Bucket</td>
			<td style="width:116px;">Plastican</td>
			<td style="width:109px;">Bway Products</td>
			<td style="width:351px;">http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Utility Fabric-Mosquito Netting White</td>
			<td style="width:116px;">Joann</td>
			<td>10173292</td>
			<td style="width:351px;">http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:209px;">Orthopedic stockings</td>
			<td>Albahealth</td>
			<td style="width:109px;">23650-040</td>
			<td style="width:351px;">product no. 081420</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Organic Raisins</td>
			<td style="width:116px;">Newman&#39;s Own</td>
			<td style="width:109px;">UPC: 884284040255</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Oviposition cups (brown)</td>
			<td>Fisher Scientific</td>
			<td>03-007-52</td>
			<td style="width:351px;">The product is actually an amber 125 mL bottle that we saw the top off of.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Recycled Paper Towels</td>
			<td style="width:116px;">Seventh Generation</td>
			<td style="width:109px;">30BPT120</td>
			<td style="width:351px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Modular Mates Square Tupperware Set</td>
			<td style="width:116px;">Tupperware</td>
			<td style="width:109px;"></td>
			<td style="width:351px;">http://order.tupperware.com/pls/htprod_www/coe$www.add_items</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Glass Grinder</td>
			<td style="width:116px;">Corning Incorporated</td>
			<td style="width:109px;">7727-2</td>
			<td style="width:351px;">These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">TRI Reagent</td>
			<td style="width:116px;">Sigma Aldrich</td>
			<td style="width:109px;">T9424</td>
			<td style="width:351px;">Apply 1ml TRI Reagent per 50-100mg of tissue. Caution - this reagent is toxic.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:209px;">TURBO DNA-free</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM1907</td>
			<td style="width:351px;">This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use.</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:209px;">RNaseZap</td>
			<td style="width:116px;">Ambion/Life Technologies</td>
			<td style="width:109px;">AM9782</td>
			<td style="width:351px;">Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">2100 Bioanalyzer</td>
			<td style="width:116px;">Agilent Technologies</td>
			<td style="width:109px;">G2939AA</td>
			<td style="width:351px;">Place up to 12 RNA samples on one chip.</td>
		</tr>
		<tr height="187">
			<td height="187" style="height:187px;width:209px;">Hemotek Membrane Feeder</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">5W1</td>
			<td style="width:351px;">This system&#160; provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company&#39;s mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:209px;">Digital Thermometer and Probe</td>
			<td style="width:116px;">Hemotek&#160;</td>
			<td style="width:109px;">MT3KFU</td>
			<td style="width:351px;">MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:209px;">Chicken Whole Blood, non-sterile with Sodium Citrate</td>
			<td style="width:116px;">Pel-Freez Biologicals</td>
			<td>33130-1</td>
			<td style="width:351px;">The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 degrees C for up to 1 year.&#160; Thaw blood at room temperature for at least 1 h before using.</td>
		</tr>
	</tbody>
</table>

an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

두 대표 RNA 샘플의 형광 측정 (그림 1A, B) NT 약 2,000 개의 밴드를 보여 주었다. 곤충, 28S 리보솜 RNA 쉽게 간단한 가열 또는 수소 결합을 방해 35 에이전트 파쇄되어 수소 결합에 의해 함께 유지 개의 폴리 뉴클레오티드 사슬로 구성된다. 생성 된 두 성분은 약 18S 리보솜 RNA와 같은 크기이다. 두 번째 RNA 샘플은 저하 높은 수준의 (그림 1B)를 보여 주었다. <p cl...

Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

아시아 타이거 모기에 Photoperiodic 휴면의 RNA-서열 분석을위한 실험 및 생물 정보학 프로토콜,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

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Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

13.2K 조회수.  Georgetown University. RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. 

비디오: An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

모스키토 리어링 (A. gambiae)에 대한 프로토콜

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

연구

생물학

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

컴퓨터 기반의 단백질 구조 및 기능 예측을위한 프로토콜

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq의 트롬빈 - 처리의 Transcriptomes 분석 및 제어 인간 폐 Microvascular 내피 세포

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

모기 내장 Metagenomic RNA-seq에 대한 Ribosomal RNA의 고갈

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

생리 녹음 및 황열병 모기의 미각 부속 기관의 RNA 시퀀싱<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

실험실 주택의 청록색 송사리 (Nothobranchius furzeri)에 대 한 프로토콜

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

무척 추 동물 모델 시스템으로 독성 테스트 실험실에서 초파리 를 사용 하 여 실험 프로토콜

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

RNA-seq를 사용하여 분자 진화 및 유전자 발현을 조사하기 위한 생물정보학 파이프라인

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

모기 전기 조영술에 대한 단계별 가이드

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...