title: GSM5729185: uMT-CDHFD-CD20-BR4; Mus musculus; RNA-Seq
library_construction_protocol: 300-1000 cells were sorted for each sample directly in a well (low binding Eppendorf) containing 5μl lysis buffer (500μl lysis buffer: 250 μl of 2X TCL buffer (Qiagen, cat.no.1070498) plus 245 μl of RNase-free water and 5 μl (1% of total volume) of b-mercaptoethanol). Nucleic acids of the lysates were purified with AMPure XP beads (Beckman Coulter). 1.8 volume of reagent and lysate were mixed thoroughly by pipette, followed and kept for 5 min at room temperature (RT). The mix was then placed onto Magnet Plate for 3 min to separate beads from the solution. The resulted cleared solution was aspirated from the reaction plate and discarded. Then were dispensed 150 μL of 80% ethanol to each well; 30 seconds at RT, aspirated out and discarded, followed by one repetition, and let it dry for 5 min at RT. Off the magnet plate, were added 6 μL of elution buffer (RNase free water), pipette and mixed 10 times. On the magnet plate 1 min to separate beads from the solution. The eluate was then transferred to the retrotranscription mix for RNA-Seq processing. For the RNA-Seq, library preparation for bulk-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016). Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3'-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. (2016)5, the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables6. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the GENCODE Version M25. RNA libraries were prepared for sequencing using standard Illumina protocols.