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Home > ÀüÁ¦Ç°º¸±â > ±â±â ¡¤ ¼Ò¸ðÇ° > NGS °ü·ÃÀåºñ > SMARTer ICELL8 single cell systemÀ¸·Î full-length single cell RNA-seq

SMARTer ICELL8 single cell systemÀ¸·Î full-length single cell RNA-seq

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º» µ¥ÀÌÅÍ´Â SMARTer ICELL8 Single-Cell SystemÀ» »ç¿ëÇÏ¿© ¼ö¹é °³ÀÇ single cell¿¡¼­ full-length sequence Á¤º¸¸¦ ¾òÀ» ¼ö ÀÖ´Â °íÇ°ÁúÀÇ RNA-seq library¸¦ »ý¼ºÇÒ ¼ö ÀÖÀ½À» º¸¿©ÁÝ´Ï´Ù. SMARTer ICELL8 systemÀ» »ç¿ëÇÑ full-length single cell RNA-seq¿¡ ´ëÇÑ ¹æ¹ýÀº cDNA ÇÕ¼º ¹× PCR ÁõÆøÀ» Æ÷ÇÔÇÏ¿© SMARTer ICELL8 chip¿¡¼­ Illumina NGS library Áغñ¿¡ ÇÊ¿äÇÑ ¸ðµç ¹ÝÀÀÀ» ¼öÇàÇÒ ¼ö ÀÖ°Ô Çϸç, chipÀ¸·ÎºÎÅÍÀÇ °á°ú¹°Àº sequencingÀÌ °¡´ÉÇÑ libraryÀÌ´Ù. SMART template-switching ±â¼úÀ» °áÇÕÇÏ¿© °íÇ°ÁúÀÇ cDNA »ý»êÀ» º¸ÀåÇÏ´Â µ¿½Ã¿¡ ´Ù¾çÇÑ single cell ºÐ¼®À» ³ôÀº °¨µµ ¹× ³ôÀº ÀçÇö¼ºÀ¸·Î ¼öÇàÇÒ ¼ö ÀÖ½À´Ï´Ù.

Full-length RNA-seq library preparation workflow

±×¸²1. SMARTer ICELL8 library preparation workflow for full-length single-cell RNA-seq.

»ì¾ÆÀÖ´Â single cellÀÌ Æ÷ÇÔµÈ well¸¸À» ¼±ÅÃÇÑ ÈÄ, cDNA´Â one-Step RT-PCR ¹ÝÀÀ(Step 2)À¸·Î oligo-dT primingÀ» ÅëÇØ ÇÕ¼ºµË´Ï´Ù. À̾îÁö´Â library Á¦ÀÛÀ» À§ÇØ ICELL8 chipÀÇ 72°³ Çà °¢°¢Àº ´Ù¸¥ index¸¦ °®°íÀÖ´Â ¸ðµç well¿¡ P5 index primers·Î index°¡ ºÐÁֵǰí, Terra polymerase¿Í reaction buffer(Step 3)°¡ ºÐÁֵ˴ϴÙ. Transposition enzyme ¹× reaction buffer(Tn5 mixture)¸¦ ¼±ÅÃµÈ well¿¡ ºÐÁÖÇÏ°í(Step 4), transposition reactionÀ» ¼öÇàÇÑ´Ù(Step 5). P7 index primer´Â well¿¡ ºÐ¹èµÇ°í, chip»óÀÇ 72°³ ¿­ °¢°¢Àº »óÀÌÇÑ index¸¦ ¹Þ°ÔµÈ´Ù(Step 6). Illumina sequencer¿¡¼­ sequencingÀÌ °¡´ÉÇϵµ·Ï ¸¸µé¾îÁø library´Â amplification(7 Step)µÇ°í chip¿¡¼­ ȸ¼öÇϸ鼭 poolingµË´Ï´Ù. pooled library´Â Illumina sequencer¿¡ loadÇϱâÀü ¼öÂ÷·Ê bead Á¤Á¦¿Í Á¤·®À» ¸¶Ä£ ÈÄ sequencing ÇÕ´Ï´Ù.

Gene-body coverage

±×¸² 2. Assessment of gene-body coverage for representative samples.

Human genome¿¡ ÀÇÇØ ÄÚµùµÈ À¯ÀüÀÚ ±¸Á¶ÀÇ ±æÀÌ°¡ Ç¥ÁØÈ­µÇ¾ú°í, »óÀÌÇÑ 2°³ÀÇ ºÐ¸®µÈ K562 cell·ÎºÎÅÍ mappingµÈ Æǵ¶ ¹üÀ§°¡ Ç¥ÁØÈ­ µÈ À¯ÀüÀÚ º»Ã¼¸¦ µû¶ó Ç÷ÎÆõǾú´Ù. (Ç¥ÁØÈ­µÈ À¯ÀüÀÚ º»Ã¼ ³»ÀÇ À§Ä¡´Â XÃà»óÀÇ ¹éºÐÀ²·Î Ç¥½ÃµÈ´Ù). corverage (YÃà¿¡¼­ 0 ´ë 1ÀÇ Ã´µµ·Î Ç¥ÇöµÊ)´Â Á¤±ÔÈ­µÈ gene body¸¦ µû¶ó °üÂûµÈ ÃÖ´ë corverage¿Í °ü·ÃÇÏ¿© Á¤±ÔÈ­µÇ¾ú´Ù.

Read-mapping distributions for data generated from single cells

±×¸² 3. Sequencing metrics for selected samples (K562 cells).

Human genomeÀÇ Ç¥½ÃµÈ ¿µ¿ª¿¡ mappingµÇ´Â readÀÇ ¹éºÐÀ²À» ³ªÅ¸³»´Â ¸·´ë ±×·¡ÇÁ(Exon(ÁÖȲ»ö), Intron(ÆĶõ»ö) ¶Ç´Â intergenic region(ȸ»ö)) histogram¿¡ °ãÃÄÁø ÁøÇÑ ÆĶõ»ö ¼±Àº °¢ well¿¡ ´ëÇØ RPKM value >0.1·Î È®ÀÎµÈ transcriptÀÇ ¼ö¸¦ ³ªÅ¸³À´Ï´Ù. °¢ histogramÀÇ ÇÏ´Ü¿¡ ÀÖ´Â ¼ýÀÚ´Â µ¥ÀÌÅ͸¦ ¾òÀº single cellÀÌ À§Ä¡ÇÑ chip»óÀÇ wellÀÇ À§Ä¡¸¦ ³ªÅ¸³À´Ï´Ù.

Relationship between read depth and number of transcripts identified

±×¸² 4. Relationship between read depth and number of transcripts identified for three single-cell containing wells.

»ó±âÀÇ ±×·¡ÇÁ´Â 3°³ÀÇ K562 cellÀÌ µé¾îÀÖ´Â well °¢°¢¿¡ ´ëÇÑ ´Ù¾çÇÑ read depths¿¡¼­ È®ÀÎµÈ transcriptÀÇ ¼ö¸¦ ³ªÅ¸³½´Ù.

Reproducibility of gene expression measurements

±×¸² 5. Reproducibility of gene expression measurements.

SMARTer ICELL8 systemÀ¸·Î »ý¼ºµÈ full-length RNA-seq µ¥ÀÌÅÍÀÇ ÀçÇö¼ºÀ» Æò°¡Çϱâ À§ÇØ, È®ÀÎ µÈ ¸ðµç transcript¿¡ ´ëÇÑ À¯ÀüÀÚ ¹ßÇö ÃøÁ¤À» HeatmapÀ¸·Î Ç¥½ÃÇÏ¿´°í, Heatmap¿¡ Ç¥½ÃµÈ °Íó·³ ¸ðµç single cellÀÌ Æ÷ÇÔµÈ well »çÀÌÀÇ Pearson correlation¸¦ °áÁ¤Çϴµ¥ »ç¿ëÇÏ¿´´Ù.